Q3 : Which cell lines do you use to localise the GFP-tagged proteins ?

Q4 : What happens when the N- / C-terminal fusions do not localise to the same structures ?

Q5 : What do you use the GFP-tagged ORFs for after they have been localised ?

Q1 : Where do the ORFs come from ?

The majority of the ORFs are derived from cDNAs identified by the cDNA consortium of the German Human Genome Project (DHGP). In addition we also GFP tag ORFs from the mammalian gene collection project (MGC).

Q2 : How do you clone so many ORFs ?

Having identified the ORF within a cDNA of interest, the ORF is first amplified by PCR, then transferred into an ‘entry vector’ using recombination cloning (Gateway from Invitrogen or Creator from BD Biosciences). This excludes any needs of screening the sequences for restriction sites. The ORF within the entry clone can then be simultaneously transferred to both the destination vectors (containing N-terminal CFP and C-terminal YFP tags) in a second recombination reaction. These final destination clones are used to determine the localisation of the encoded protein.

Q3 : Which cell lines do you use to localise the GFP-tagged proteins ?

The initial localisation screen is made in Vero cells - a monkey kidney fibroblast line. These cells are used since they are particularly flat, with a large area, therefore enabling accurate determination of localisation to be made. From experience we know that localisations in these cells are >98% similar to those observed in human cell lines. Particularly interesting ORFs are also screened for localisation in PC12 cells and hippocampal neurons.

Q4 : What happens when the N- / C-terminal fusions do not localise to the same structures ?

A decision about localisation is always made with respect to the bioinformatic information we have about the ORF. In addition, localisation to distinct membrane structures is almost always considered more significant than a more diffuse localisation pattern. The scheme below shows how the final decision is made.

Q5 : What do you use the GFP-tagged ORFs for after they have been localised ?

In the Pepperkok lab the ORFs are screened in a series of functional assays for any potential role in membrane traffic - the main focus of the lab. In addition we have numerous collaborators who use this GFP-ORF ‘library’ in their own screens to look for candidates that may be involved in the particular biological processes in which they are interested.